Download Technical standards and guidelines for spinal muscular atrophy testing

ACMG POLICY STATEMENTS, STANDARDS
AND
GUIDELINES
Technical standards and guidelines for spinal muscular
atrophy testing
Thomas W. Prior, PhD1, Narasimhan Nagan, PhD2, Elaine A. Sugarman, MS, CGC2,
Sat Dev Batish, PhD3, and Corey Braastad, PhD3
Disclaimer: These ACMG Standards and Guidelines are developed primarily as an educational resource for
clinical laboratory geneticists to help them provide quality clinical laboratory genetic services. Adherence to
these standards and guidelines is voluntary and does not necessarily assure a successful medical outcome. These
Standards and Guidelines should not be considered inclusive of all proper procedures and tests or exclusive of
other procedures and tests that are reasonably directed to obtaining the same results. In determining the
propriety of any specific procedure or test, the clinical laboratory geneticist should apply his or her own
professional judgment to the specific circumstances presented by the individual patient or specimen.
Clinical laboratory geneticists are encouraged to document in the patient’s record the rationale for the use
of a particular procedure or test, whether or not it is in conformance with these Standards and Guidelines. They
also are advised to take notice of the date any particular standard or guidelines was adopted, and to consider
other relevant medical and scientific information that becomes available after that date. It also would be prudent
to consider whether intellectual property interests may restrict the performance of certain tests and other
procedures.
Abstract: Spinal muscular atrophy is a common autosomal recessive
neuromuscular disorder caused by mutations in the survival motor neuron
(SMN1) gene, affecting approximately 1 in 10,000 live births. The disease
is characterized by progressive symmetrical muscle weakness resulting
from the degeneration and loss of anterior horn cells in the spinal cord and
brainstem nuclei. The disease is classified on the basis of age of onset and
clinical course. Two almost identical SMN genes are present on 5q13: the
SMN1 gene, which is the spinal muscular atrophy-determining gene, and
the SMN2 gene. The homozygous absence of the SMN1 exon 7 has been
observed in the majority of patients and is being used as a reliable and
sensitive spinal muscular atrophy diagnostic test. Although SMN2 produces
less full-length transcript than SMN1, the number of SMN2 copies has been
shown to modulate the clinical phenotype. Carrier detection relies on the
accurate determination of the SMN1 gene copies. This document follows
the outline format of the general Standards and Guidelines for Clinical
Laboratories. It is designed to be a checklist for genetic testing professionals who are already familiar with the disease and methods of analysis.
Genet Med 2011:13(7):686 – 694.
From the 1Ohio State University, Columbus, Ohio; 2Genzyme Genetics, Westborough, Massachusetts; and 3Athena Diagnostics, Worcester, Massachusetts.
Thomas W. Prior, PhD, Department of Pathology, Ohio State University,
125 Hamilton Hall, 1645 Neil Ave, Columbus, OH 43210. E-mail:
[email protected].
Disclosure: N.N. and E.A.S. are employees of Genzyme Genetics. S.D.B.
and C.B. are employees of Athena Diagnostics. Both of these companies
perform laboratory testing for SMA. T.W.P. does not have a commercial interest
in SMA testing but does perform the testing in his laboratory. In addition, he has
received generous research funding from the Claire Altman Heine Foundation,
Inc. Genzyme Genetics and its logo are trademarks of Genzyme Corporation and
used by Esoterix Genetic Laboratories, LLC, a wholly owned subsidiary of
LabCorp, under license. Esoterix Genetic Laboratories and LabCorp are operated independently from Genzyme Corporation.
Published online ahead of print June 13, 2011.
DOI: 10.1097/GIM.0b013e318220d523
686
Key Words: spinal muscular atrophy, survival motor neuron, SMN1,
SMN2, genotype, phenotype, genetic testing, carrier testing
D
isease-specific statements are intended to augment the current general American College of Medical Genetics
(ACMG) Standards and Guidelines for Clinical Genetic Laboratories. Individual laboratories are responsible for meeting the
CLIA/College of American Pathologists (CAP) quality assurance standards with respect to appropriate sample documentation, assay validation, general proficiency, and quality control
measures.
BACKGROUND ON SPINAL MUSCULAR
ATROPHY
Gene symbol/chromosome locus
Survival motor neuron 1 (SMN1) gene at chromosome
5q11.2-13.3.
OMIM #
Spinal muscular atrophy (SMA) type I (253300), SMA type
II (253550), and SMA type III (253400).
Brief clinical description
The autosomal recessive disorder proximal SMA is a severe
neuromuscular disease characterized by degeneration of alpha
motor neurons in the spinal cord, which results in progressive
muscle weakness and paralysis. The predominant pathologic
feature on autopsy studies of patients with SMA is loss of motor
neurons in the ventral horn of the spinal cord and in brainstem
motor nuclei. SMA is the second most common fatal autosomal
recessive disorder after cystic fibrosis, with an estimated incidence of approximately 1 in 10,000 live births.1 Childhood
SMA is subdivided into three clinical groups on the basis of age
of onset and clinical course.2,3 Type I SMA (Werdnig-Hoffman
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disease) is characterized by severe, generalized muscle weakness and hypotonia at birth or within the first 3 months. Death
from respiratory failure usually occurs within the first 2 years.
Approximately 60 –70% of patients with SMA have the type I
disease.4 Type II children are able to sit, although they cannot
stand or walk unaided, and typically survive beyond 4 years.
The phenotypic variability exceeds that observed in type I
patients, ranging from infants who sit transiently and demonstrate severe respiratory insufficiency to children who can sit,
crawl, and even stand with support. Prognosis in this group is
largely dependent on the degree of respiratory involvement.
Type III SMA (Kugelberg-Welander disease) is a milder form,
with onset during infancy or youth: patients learn to walk
unaided and have prolonged survivals. They comprise a less
fragile group than type II patients with respect to respiratory and
nutritional vulnerability. Type III SMA is further subdivided
into two groups: type IIIa (onset before 3 years of age) and type
IIIb (onset at age ⱖ3 years). Cases presenting with the first
symptoms of the disease at the age of 20 –30 years are classified
as type IV or proximal adult type SMA. The described classification is based on age of onset and clinical course, but it
should be recognized that the disorder demonstrates a continuous range of severity. For more information, see the online
Gene Reviews profile at http://www.ncbi.nlm.nih.gov/books/
NBK1352/.
Mode of inheritance
Inheritance is autosomal recessive, with variable expression.
Gene description/normal gene product
The SMA gene is within a complex region containing multiple repetitive and inverted sequences.5 The SMN gene (Entrez
Gene ID number 6606) comprises nine exons with a stop codon
present near the end of exon 7.6 Two inverted SMN copies are
present: the telomeric or SMN1 gene, which is the SMA-determining gene and the centromeric or SMN2 gene. The two SMN
genes are highly homologous, have equivalent promoters, and
only differ at five base pairs.5,7,8 The base differences are used
to differentiate SMN1 from SMN2. The coding sequence of
SMN2 differs from that of SMN1 by a single nucleotide
(840C⬎T), which does not alter the amino acid but has been
shown to be important in splicing. SMA results from a reduction
in the amount of the SMN protein, and there is a strong
correlation between the disease severity and SMN protein levels.9,10 The SMN protein is a ubiquitously expressed, highly
conserved 294-amino acid polypeptide. The protein is found in
both the cytoplasm and nucleus and is concentrated in punctate
structures called “gems” in the nucleus. High levels of the
protein have been found to exist in the spinal motor neurons, the
affected cells in patients with SMA. The protein self-associates
into multimeric structures. Biochemically, SMN does not seem
to exist within cells in isolation but instead forms part of a large
protein complex, the SMN complex. Many of these SMN interacting proteins are components of various ribonuclear protein
(RNP) complexes that are involved in distinct aspects of RNA
metabolism. The best characterized function of the SMN complex is regulating the assembly of a specific class of RNAprotein complexes, the small nuclear RNPs (snRNPs).11,12 The
snRNPs are a critical component of the spliceosome; a large
RNA protein that catalyzes premRNA splicing. SMA may,
therefore, be a disorder resulting from aberrant splicing. As the
SMN protein is ubiquitously expressed, it remains unknown
how a loss of a general housekeeping function (snRNP assembly) causes a selective loss of motor neurons in SMA.13 The
high expression of SMN protein in motor neurons may suggest
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SMA standards and guidelines
that the neuronal population is more sensitive to decreases in the
SMN protein level. Possibly, the altered splicing of a unique set
of premessenger RNAs results in deficient proteins, which are
necessary for motor neuron growth and survival. In addition to
its role in spliceosomal ribonucleoprotein assembly, SMN may
have other functions in motor neurons. A subset of SMN complexes is located in axons and growth cones of motor neurons
and may be involved in some aspects of axonal transport and
localized translation of specific messenger RNAs.14,15
The SMN1 mutation
Homozygous mutations of the SMN1 gene cause SMA. Both
copies of the SMN1 gene are absent in approximately 95% of
affected patients, whereas the remaining patients have nonsense, frameshift, or missense mutations within the gene.16
Based on Hardy-Weinberg equilibrium, the remaining patients
(with the smaller types of mutations) are virtually all assumed to
be hemizygous for the SMN1 deletion. The absence of SMN1
can occur by deletion, typically a large deletion that includes the
whole gene or by conversion to SMN2. Although patients with
SMA have mutations in SMN1, they always carry at least one
normal copy of SMN2, which is partially functional but unable
to fully compensate for the deficiency of the SMN1 protein. The
homozygous loss of both genes has not been reported, presumably as a result of lethality.
Genotype/phenotype association
SMN1 exon 7 is absent in the majority of patients independent of the severity of SMA. Several studies have shown that
the SMN2 copy number modifies the severity of the disease.17–20
The SMN2 copy number varies from 0 to 3 copies in the normal
population, with approximately 10 –15% of normal individuals
having no SMN2. However, patients with a milder phenotype
with type II or III SMA have been shown to often have more
copies of SMN2 than type I patients. The majority of patients
with the severe type I form have one or two copies of SMN2;
most patients with type II have three SMN2 copies; and most
patients with type III have three or four SMN2 copies. Three
unaffected family members of patients with SMA, with confirmed SMN1 homozygous deletions, were shown to have five
copies of SMN2.21 These cases not only support the role of
SMN2 modifying the phenotype but they also demonstrate that
expression levels consistent with five copies of the SMN2 genes
may be sufficient to compensate for the absence of the SMN1
gene. This inverse dose-relationship between SMN2 copy number and disease severity has also been supported by the SMA
mouse model.22,23 The SMA mouse models have not only
confirmed the susceptibility of motor neuron degeneration to
SMN deficiency but have also verified that the degeneration can
be prevented by increased SMN2 dosage. Mice lacking the
endogenous mouse SMN gene but expressing two copies of the
human SMN2 gene develop severe SMA and die within 1 week
of age; however, mice that express multiple copies of SMN2 do
not develop the disease. In addition to the SMN2 copy number,
other modifying factors influence the phenotypic variability of
SMA. There are very rare families reported in which markedly
different degrees of disease severity are present in affected
siblings with the same SMN2 copy number. These discordant
sib pairs, which share the same genetic background around the
SMA locus, would indicate that there are other modifier genes
outside the SMA region. Differences in splicing factors may
allow more full-length expression from the SMN2 gene and
account for some of the variability observed between discordant
sibs.24 It was also found that in some rare families with unaffected SMN1-deleted females, the expression of plastin 3 was
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higher than in their SMA affected counterparts.25 Plastin 3 was
shown to be important for axonogenesis and, therefore, may act
as a protective modifier.
Mutational mechanism
The SMN1 gene produces full-length transcript, whereas the
SMN2 gene produces predominantly an alternatively spliced
transcript (exon 7 deleted) encoding a protein (SMN⌬7) that
does not oligomerize efficiently and is unstable.26,27 The inclusion of exon 7 in SMN1 transcripts and exclusion of this exon in
SMN2 transcripts are caused by the single nucleotide difference
at ⫹6 in SMN1 exon 7 (c.840C⬎T). Although the C to T change
in SMN2 exon 7 does not change an amino acid, it does disrupt
an exonic splicing enhancer (ESE) or creates an exon silencer
element (ESS) that results in the majority of transcripts lacking
exon 7.28,29 The ESEs and ESSs are cis-acting exonic sequences
that influence the use of flanking splice sites. ESEs stimulate
splicing and are often required for efficient intron removal,
whereas ESSs inhibit splicing. Whether it is the loss of an ESE
or creation of an ESS, the result is a reduction of full-length
transcripts generated from SMN2. A single SMN2 gene produces less functional protein compared with a single SMN1
gene.9,10,20 –30 Therefore, SMA arises because the SMN2 gene
cannot fully compensate for the lack of functional SMN when
SMN1 is mutated. However, small amounts of full-length transcripts generated by SMN2 are able to produce a milder type II
or III phenotype when the copy number of the SMN2 gene is
increased. SMA is, therefore, caused by low levels of SMN
protein, rather than a complete absence of the protein. A recent
report described three unrelated patients with SMA who possessed SMN2 copy numbers that did not correlate with the
observed mild clinical phenotypes.31 A single base substitution
in SMN2, c.859G⬎C, was identified in exon 7 in the patients
DNA, and it was shown that the substitution created a new ESE
element. The new ESE increased the amount of exon 7 inclusion
and full-length transcripts generated from SMN2, thus resulting
in the less severe phenotypes. Therefore, the SMA phenotype
may not only be modified by the number of SMN2 genes but
SMN2 sequence variations can also affect the disease severity.
It should, therefore, not be assumed that all SMN2 genes are
equivalent and sequence changes found within the SMN2 gene
must be further investigated for potential positive or negative
effects on SMN2 transcription when there is a lack of correlation
between the genotype and phenotype.
Listing of mutations
Although the absence of both copies of the SMN1 gene is a
very reliable and sensitive assay for the molecular diagnosis of
SMA, approximately 5% of affected patients have other types of
mutations in the SMN1 gene that will not be detected by
homozygous deletion testing. Because of the high deletion
frequency and according to the Hardy-Weinberg equilibrium,
most of these patients will be compound heterozygotes; with
one SMN1 allele being deleted and the other allele with a point
mutation or other types of small mutations. If a patient with
SMA possesses only a single copy of SMN1, it is likely that the
remaining copy contains a more subtle mutation, including
nonsense mutations, missense mutations, splice site mutation
insertions, and small deletions. Many of the same intragenic mutations have now been reported in unrelated patients.19,32,33 The
most frequently reported mutations are the p.Tyr272Cys (c.815A⬎G),
c.399_402delAGAG, c.770-780dup11, and p.Thr274Ile (c.821C⬎T).
The coding region for SMN can be found on ensemble (www.
ensemble.org). Proper nomenclature must be used to report any
verified sequence mutation (http://www.hgvs.org/mutnomen/).
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Table 1 Diagnostic mutation categories
Mutation category
% Affected
Genotype designation
关0 ⫹ 0兴 or a2
Category 1
95
Category 2
5
关1d ⫹ 0兴 or 2ad
Category 3
Rare
关1d ⫹ 1d兴 or d2
Ethnic association of SMA
With an incidence of approximately 1 in 10,000 live births
and a carrier frequency of 1/40 –1/60, SMA is the leading
inherited cause of infant mortality. A recent report provides
carrier frequencies in several populations including white, Ashkenazi Jewish, African American, Asian, and Hispanic.34 The
lower carrier frequencies found in African Americans and Hispanics would suggest a lower prevalence of SMA in these
populations.
GUIDELINES
Definition of normal and mutation category
Alleles
Among normal alleles, a chromosome with one copy of
SMN1 gene is designated as a “1-copy” or “1” allele. A chromosome bearing two copies of SMN1 gene is designated as
a “2-copy” or “2” allele. The most common disease allele bears
a deletion (or gene conversion of SMN1 to SMN2) resulting
in a loss of exon 7 (⌬7SMN1) is referred to as the “0-copy” or
“0” allele. Disease alleles with subtle intragenic point mutations
on the SMN1 gene are referred to as 1d. The 0, 1, 2, and 1d
alleles are also variably referred to as a, b, c, and d alleles,
respectively, in literature.35 The resulting allele pairings that
give rise to the diagnostic and carrier genotypes of SMA are
defined later.
Diagnostic
The term “diagnostic” in the context of SMA is characterized
by the presence of mutations classified into either of three
categories (Table 1). Category 1, which accounts for approximately 95% of affected individuals, is characterized by a homozygous absence of SMN1 exon 7 (⌬7SMN1) due to deletions
or gene conversions of SMN1 gene to SMN2 and is designated
as the [0 ⫹ 0] genotype or a2.36 The resulting diagnostic finding
is the absence of detectable SMN1 exon 7 and SMN1 exon 7
copy number of 0. Category 2, which accounts for approximately 5% of affected individuals, is characterized by compound heterozygosity for a rare intragenic point mutation within
the SMN1 gene on one chromosome and a deletion/gene conversion of SMN1 exon 7 (⌬7SMN1) on the other chromosome
and is designated as the [1d ⫹ 0] genotype or 2ad. The resulting
diagnostic finding may be a detectable level of SMN1 exon 7
and an SMN1 exon 7 copy number of either 0 or 1 depending on
the location of the rare intragenic point mutation within the
SMN1 gene and its ability to interfere with SMN1 copy number
analysis. Category 3, which is very rare and is most likely due
to consanguinity, is characterized by subtle intragenic point
mutations within the SMN1 gene on both chromosomes and
is designated as the [1d ⫹ 1d] genotype or d2. The resulting
diagnostic finding may be a detectable level of SMN1 exon 7
and an SMN1 exon 7 copy number of 0, 1, or 2 depending on
the location of each rare intragenic point mutation within the
© 2011 Lippincott Williams & Wilkins
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SMN1 gene and its ability to interfere with SMN1 copy
number analysis.
senting with symptoms of proximal muscle weakness, fasciculations, dysphagia, dysarthria, and absent deep tendon reflexes.
Carrier
Sensitivity and specificity
The analytical sensitivity of SMN1 deletion/copy number
analysis (proportion of homozygous ⌬7SMN1 among all mutations in diagnostic category 1) is ⬎99% (using the dosage
assays described in the guidelines). The clinical sensitivity
(proportion of homozygous ⌬7SMN1 if 5q13-linked SMA is
present) of the diagnostic test is approximately 95%. The remaining 5% of patients fall into either of diagnostic categories
2 or 3 (i.e., the [1d ⫹ 0] and the [1d ⫹ 1d] genotypes) and
represent a source of false-negative diagnostic test results.3
These are patients with other types of small mutations within
the SMN1 gene and will not be detected by the deletion testing.
Although the absence of both copies of the SMN1 gene is a
very reliable and sensitive assay for the molecular diagnosis of
SMA, approximately 5% of affected patients have other types of
mutations in the SMN1 gene that will not be detected by
homozygous deletion testing. Because of the high deletion
frequency and according to the Hardy-Weinberg equilibrium,
most of these patients will be compound heterozygotes; with
one SMN1 allele being deleted and the other allele with a point
mutation or other types of small mutations. If a patient with an
SMA-like phenotype possesses only a single copy of SMN1, it
is likely that the remaining copy contains a more subtle mutation, including nonsense mutations, missense mutations, splice
site mutation insertions, and small deletions.
Both the analytical specificity (proportion of negative test
results if homozygous ⌬7SMN1 SMA genotype is not present)
and the clinical specificity (proportion of negative test results if
5q13-linked SMA is not present) of SMN1 deletion/copy number analysis are ⬎99%. Polymorphisms or point mutations
under the primer and/or the probe binding regions may influence the analytical and clinical specificity by increasing the
false-positive rate depending on the technology used.37 As a
measure of additional quality assurance, follow-up sequencing
underneath the primer and probe binding regions on all diagnostic (0 or 1 SMN1 copy number) results is expected to rule out
a false-positive diagnostic finding attributable to this phenomenon and provide a better understanding of the underlying
molecular mechanism of the mutation identified.
De novo deletion or gene conversions of paternal origin have
been reported to occur at a frequency of 2% of patients with
SMA.38 Carrier testing on both parents of patients with homozygous ⌬7SMN1 SMA may provide additional information on the
occurrence of de novo deletions. If one of the parents seems to
be a noncarrier of SMA (i.e., ⱖ2 copies of SMN1), further
carrier testing on both parents of the noncarrier parent (i.e., the
grandparents) can be pursued to determine the phase of SMN1
genes in the noncarrier parent. This helps distinguish a [1 ⫹ 1]
genotype leading to a de novo deletion in the index case, from
a [2 ⫹ 0] obligate carrier genotype in the seemingly noncarrier
parent. Furthermore, germline mosaicism for SMN1 deletion/
gene conversions has been reported.6 Although the detection of
a de novo mutation in an SMA family substantially lowers the
recurrence risk, prenatal diagnosis in subsequent pregnancies
should still be considered due to the rare possibility of a recurrent de novo mutation or germ-line mosaicism leading to an
affected child.
The term “carrier” in the context of SMA is classified into
either of three categories (Table 2). Category 1 is characterized
by an SMN1 exon 7 copy number of 1 and presumes the
presence of an SMN1 deletion/gene conversion on the other
chromosome (heterozygous ⌬7SMN1) and is designated as the
[1 ⫹ 0] genotype or 2ab. Category 2 is characterized by a
presence of two SMN1 genes in cis on a single chromosome
along with a deletion/gene conversion of SMN1 exon 7 on the
opposite chromosome resulting in an SMN1 exon 7 copy number of 2 and is designated as the [2 ⫹ 0] genotype or 2ac.
Category 3 is characterized by a subtle intragenic point mutation on one chromosome resulting in an SMN1 exon 7 copy
number of ⱖ2 and is designated as the [1 ⫹ 1d], and [2 ⫹ 1d]
genotypes or 2bd and 2cd, respectively. It must be recognized
that other rare carrier genotypes such as [3 ⫹ 0] or [3 ⫹ 1d] are
likely and are thought to occur at a lower frequency relative to
the most common carrier genotypes.36
Negative result
A negative test result is characterized by the presence of
detectable amounts of SMN1 exon 7, with an SMN1 exon 7 copy
number of ⬎1, with the presence of subtle intragenic point
mutations within the SMN1 gene having been ruled out. If the
presence of subtle intragenic mutations has not been ruled out,
a negative test result decreases the likelihood but does not
exclude the diagnosis of SMA. Within the context of carrier
testing, an SMN1 copy number of ⱖ2 is associated with a
reduced risk to be a carrier.
TESTING CONSIDERATIONS
As with any genetic testing modality, the required intake
information needed to facilitate an accurate result interpretation
includes the reason for referral, i.e., diagnostic versus carrier
testing, an accurate representation of family history, i.e., additional affected or carrier individuals identified within the family,
clinical or diagnostic findings of relevance to SMA, and patient
ethnicity. Patient ethnicity helps to provide appropriate risk
assessment after a negative carrier testing result in an individual
with no family history of SMA.
Diagnostic mutation analysis
Testing by SMN1 deletion or copy number analysis is indicated for individuals with a suspected diagnosis of SMA, pre-
Table 2 Commonly designated carrier mutation
categories
Mutation category
Category 1
SMN1 exon 7
copy number
Genotype
designation
1
关1 ⫹ 0兴 or 2ab
2
关2 ⫹ 0兴 or 2ac
a
2
关1 ⫹ 1d兴 or 2bd
Category 3a
ⱖ2
关2 ⫹ 1d兴 or 2cd
Category 2
Category 3
a
SMN1 exon 7 copy number in Category 3 may depend on the location of the point
mutation within the SMN1 gene and its ability to interfere with copy number
analysis.
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Carrier testing
Carrier testing for SMA should be offered to asymptomatic
individuals with a confirmed or suspected family history of
SMA. Given the 1/40 –1/60 carrier frequency of SMA, popula689
Prior et al.
tion carrier screening has recently been recommended by the
ACMG39 but has not been supported by the American College
of Obstetricians and Gynecologists’ Committee on Genetics.40
Carrier frequencies determined from derived allele frequencies
should consider all combinations of the most likely allele pairings expected to occur among carriers in the general population,
namely [1 ⫹ 0], [1 ⫹ 1d], [2 ⫹ 0], and [2 ⫹ 1d]. Carrier
frequencies derived solely from the observed 1-copy genotype
frequencies tend to underestimate the prior risk estimates for
carriers of SMA in the general population, thereby leading to
inaccuracies in posterior risk estimates after carrier testing. The
report by Ogino and Wilson35 provides Bayesian risk calculations for several situations.
The sensitivity (true positives/true positives ⫹ false negatives, or the detection rate) of the carrier test is defined as the
proportion of carriers with the heterozygous ⌬7SMN1, 1-copy
genotype among all SMA carriers (including point mutation
carriers and deletion carriers with two copies of the SMN1 gene)
and can be expressed as the ratio [1 ⫹ 0]/[1 ⫹ 0] ⫹ [1 ⫹ 1d]
⫹ [2 ⫹ 0] ⫹ [2 ⫹ 1d]. Here a subset of the denominator, [1 ⫹
1d] ⫹ [2 ⫹ 0] ⫹ [2 ⫹ 1d], represents a source of false-negative
carrier test results. Detection rate varies by ethnicity ranging
from 71% in African Americans to 95% in whites.34 The major
contributor to this ethnicity-based variation in detection rate is
the occurrence of two (or more) SMN1 genes in tandem on a
single chromosome 5 (i.e., the [2 ⫹ 0] Category 2 carrier
genotype). The estimated frequency of alleles with two or more
copies of SMN1 is 3– 8 times more prevalent in African Americans, when compared with other ethnic groups.34 This translates to a much higher frequency of individuals with the SMA
carrier [2 ⫹ 0] genotype among African Americans compared
with other races. It also has important implications in risk
assessment and counseling after carrier screening in individuals
of African American ancestry.
As with the diagnostic test, analytical specificity of the carrier test (proportion of negative test results if not a carrier) is
⬎99% if the presence of polymorphisms underneath the primer
and/or probe binding sites are ruled out. The negative predictive
value, i.e., the proportion of negative tests that correctly identifies noncarriers (true negatives/true negatives ⫹ false negatives), is ⬎99% regardless of ethnicity. The positive predictive
value, i.e., the proportion of positive tests that correctly identify
carriers of 5q13 linked SMA (true positive/true positive ⫹ false
positives) is ⬎99% if the presence of polymorphisms underneath the primer and/or probe binding sites are ruled out.
A negative test result within the context of carrier screening
is defined by an SMN1 exon 7 copy number ⱖ2 with a reduced
posterior risk for being a carrier. The posterior risk determination takes into consideration the conditional probability of being
either a carrier or a noncarrier after an SMN1 copy number
analysis result of ⱖ2.36,41 The posterior residual risk for an
individual with no family history of SMA to be a carrier
following the identification of two copies of SMN1 exon 7
varies by ethnicity ranging from 1:632 for an individual of
white ancestry to 1:121 for an individual of African American
ancestry.34 The residual risk following identification of three
copies of SMN1 exon 7 is considerably lower and varies from
1:3500 for an individual of white ancestry to 1:3000 for an
individual of African American ancestry.34 As with other carrier
screening tests, patients need to understand that a negative
carrier screen reduces but does not eliminate the risk to be a
carrier of SMA. Risk assessment calculations using Bayesian
analysis are essential for the proper genetic counseling of SMA
families.35
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The laboratory must establish validated, nonoverlapping cutoff values that can accurately and reliably distinguish SMN1
copy numbers of 0, 1, 2, and ⱖ3. The accuracy, precision, and
confidence of SMN1 copy number measurements around these
established cutoff values should be known to the laboratory.
Copy number variations within the genomic internal standard
and inefficiency of polymerase chain reaction (PCR) amplification of the internal standard reference gene relative to the SMN1
gene represent additional sources of false positive or incorrect
copy number estimates. Therefore, the copy number of genomic
internal standard reference gene should be constant at two
copies within the genome, and the PCR amplification efficiency
of the SMN1 gene relative to the chosen internal reference
standard gene should be consistent between analyses. Performing replicate copy number measurements with two independent
two copy number internal standard reference genes can help
assure the accuracy of copy number analysis.
Prenatal testing
Indications for prenatal diagnosis of SMA include a 25% risk
for the fetus to be affected (when both carrier parents are
identified as a result of family history or following carrier
identification by population screening) or the presence of abnormal findings such as decreased fetal movements and contractures in utero or increased NT on fetal ultrasound. Prenatal
testing for SMA is performed by direct determination of the
homozygous exon 7 deletion. A prerequisite is the previous
identification of the homozygous deletion in the index case or
positive carrier status in the parents. Testing both parents and
the prenatal specimen by the same methodology, in addition to
ruling out false-positive results attributed to sequence variants
underneath the primer and probe binding sites, facilitates the
most accurate interpretation of the prenatal test result. As maternal cell contamination of the fetal specimen can result in a
false-negative test result, such contamination must be concurrently ruled out before reporting the prenatal test result. Although the presence of homozygous ⌬7SMN1 is consistent with
a diagnosis of SMA, the clinical severity, i.e., the type of SMA,
cannot be predicted based on these molecular results.
Linkage
In families where a sample from a previous affected child is
available, linkage analysis using microsatellite markers flanking
the SMN1 gene may be considered when SMN1 deletion analysis is negative, and the presence of subtle intragenic mutations
tracking within the family is suspected. Another indication for
linkage analysis is in distinguishing a [1 ⫹ 1] genotype from a
[2 ⫹ 0] genotype, when two copies of SMN1 are identified in
the parent of an affected child with a homozygous ⌬7SMN1. In
this latter scenario, linkage analysis can also identify recombination events associated with de novo deletions occurring
within the SMN1 gene.
SMN2 copy number
SMN2 copy number analysis is not routinely performed
within the setting of diagnostic or carrier testing for SMA.
SMN2 copy number analysis may be of value within the setting
of clinical trials and newborn screening in stratifying patients
who are more likely to respond to therapeutic strategies aimed
at upregulating the levels of expression of full-length SMN
protein from the SMN2 gene. Within clinical or prenatal diagnostic settings, however, results from SMN2 copy number analysis if available must be interpreted with caution. Although
SMN2 copy number influences disease severity, SMN2 sequence
variants and other genes have also been implicated in influenc© 2011 Lippincott Williams & Wilkins
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ing SMA phenotype.42 Therefore, SMN2 copy number results
may provide probabilistic information regarding clinical severity for an affected child or fetus but should not be viewed as
definitive. Furthermore, an SMN2 copy number result indicates
total SMN2 copy number for both alleles; therefore, it is not
possible to determine SMN2 phase in unaffected individuals.
For indications of carrier testing, SMN2 copy number determination does not provide information useful for counseling. For
example, an SMN1 deletion carrier ([1 ⫹ 0] genotype) may
carry three copies of SMN2, but it is not possible to determine
how many of those SMN2 copies are in cis with the deletion and
would, therefore, be passed to offspring. An ability to determine
phase is not clinically available at this time but would be
necessary to add value to determination of SMN2 copy number
in carrier parents.
METHODOLOGICAL CONSIDERATIONS
Individual US laboratories offering molecular diagnostic and
carrier testing for SMA should be in compliance with all federal
and state regulations relevant to clinical laboratory operations.
This includes meeting all CLIA/CAP quality control requirements. In addition, all laboratories should be active participants
in annual CAP SMA proficiency testing challenges. All methodological applications should also be in compliance with the
current Standards and Guideline for Clinical Genetics Laboratories developed by the Laboratory Practice Committee of the
ACMG. Non-US laboratories should be similarly compliant
with their individual countries statutory regulations governing
oversight of clinical laboratories.
The absence of detectable SMN1 in patients with SMA is
being used as a reliable and powerful diagnostic test for the
majority of patients with SMA. The first diagnostic test for a
patient suspected to have SMA should be the SMN1 gene
deletion test. Both copies of the SMN1 exon 7 are absent in
approximately 95% of affected patients, whereas small more
subtle mutations have been identified in the remaining affected
patients. The molecular diagnosis of the SMA consists of the
detection of the absence of exons 7 of the SMN1 gene. Genetic
testing is not only the most rapid and sensitive method to
confirm the diagnosis but also the testing allows for further
invasive investigations such as electromyography and muscle
biopsy to be avoided. SMN1 dosage testing is used to determine
the SMN1 copy number and detect SMA carriers: carriers will
possess one SMN1 copy and noncarriers will have two SMN1
copies and occasionally have three SMN1 copies. There are a
number of methods being used for the determination of SMN1
copy number, with multiplex ligation-dependent probe amplification (MLPA) and quantitative PCR (qPCR) being the most
common. SMN1 sequencing is used for the identification of the
compound heterozygote affected state. All general guidelines
for PCR, restriction fragment length polymorphism (RFLP),
MLPA, qPCR, and DNA sequencing in the ACMG Standards
and Guidelines apply. These technologies have limitations and
strengths when applied to diagnostic and/or carrier testing for
SMA as detailed later.
RFLP test
The RFLP test is commonly used and allows for the detection
of the homozygous deletion of SMN1 exon 7. It is currently
being used as an assay for the diagnosis of SMA in both clinical
and prenatal settings.43 The PCR primer sets first amplify both
SMN1 and SMN2 exon 7. Although this is a highly repetitive
region, the exon 7 base pair difference (840C⬎T) alters a DraI
restriction enzyme site (due to a mismatched primer) and allows
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SMA standards and guidelines
one to easily distinguish SMN1 from SMN2 on digestion of the
PCR products. The absence of the undigested SMN1 exon 7
product is consistent with the SMA diagnosis. Presence of an
undigested product band indicates one or more SMN1 copies.
Another restriction enzyme that can be used in the RFLP assay
is HinfI.19 In the HinfI assay, internal control restriction sites are
introduced, which allows for the assessment of complete digestion. Regardless of the enzyme used, the appropriate positive
and negative controls should always be included in every assay.
Technical advantages of SMA RFLP include (1) reliability of
the assay, (2) very robust and minimally sensitive to DNA
quality or degradation, and (3) simplicity to set up and operate
in a clinical diagnostic laboratory. Technical disadvantages of
SMA RFLP include (1) the need to avoid partial digestion
problems and (2) DNA sequence variants located under the
SMN1 primer binding sites or enzymatic restriction site that
may prevent the primers from annealing properly or proper
digestion and might yield a false-positive diagnostic (zero copies) result. Clinical disadvantages of SMA RFLP include the
inability to detect carrier status and determine SMN2 copy
number.
Multiplex ligation-dependent probe amplification
MLPA is a PCR-based method of quantifying multiple
genomic loci in a single reaction. It is based on the ligation of
a set of two oligonucleotides probes that have annealed adjacently to a target sequence. Only ligated probes can serve as a
template for a subsequent PCR. MLPA analysis of DNA from
different individuals should reproducibly generate each of the
expected peaks, and their sizes should correspond to those listed
for each probe pair. MLPA technology is able to detect copy
number of specific genomic loci and, therefore, can be used to
test for SMA diagnostic and carrier status in clinical and prenatal settings.44 Probe sets specific to exon 7 of SMN1, SMN2
(MRC Holland SALSA MLPA KIT P021-A1 SMN Exon 7
probes 1260-L0966 and 1260-L0967), and 20 typically diploid
loci located throughout the genome are used in SMA MLPA.
After performing the enzymatic reactions, the PCR products are
run through a capillary electrophoresis (CE) analyzer. The relative intensities and morphologies of the peaks should be consistent from one sample to the next. When the larger amplification products are weaker than the smaller fragments, this
usually indicates that PCR amplification was not optimal or that
the DNA sample analyzed might be degraded. Computer-aided
scoring is a sensitive method to normalize the peak height or
area for each PCR product compared with the 20 endogenous
control loci. The median ratios across all the samples for each
probe can be used as a reference value. Reference values for a
copy number of 2 should approximately 1. A heterozygous
deletion should give a ratio of approximately 0.5, whereas an
elevated copy number should give a reference value ⬎1.5.
Reference values for a homozygous deletion (SMA positive) are
close to 0. MLPA technology has several key advantages,
including (1) allowance for simultaneous detection of SMN1
and SMN2 copy numbers, therefore, helping differentiate SMA
type 1 from SMA types 2 and 3, (2) a high degree of precision
for the quantitative detection of three or fewer SMN1 copies, (3)
20 independent control loci are able to be assayed in one
reaction, (4) all reactions are performed in a single tube, and (5)
probe sets are easy to obtain commercially, (6) a high degree of
reproducibility and a large number of samples can be tested
simultaneously, and (7) only 20 ng of genomic DNA is required, but 5– 6-fold more DNA can be successfully technically
validated. MLPA technology has several important limitations
including (1) DNA sequence variants located under probe bind691
Prior et al.
ing sites of SMN1 alleles may interfere with probe hybridization
and might result in a false-positive carrier (one copy) or falsepositive diagnostic (zero copies) result, (2) reactions are sensitive to contaminants but generate uninterpretable results, (3)
MLPA cannot yet be used to investigate single cells, which is
important for preimplantation genetic diagnosis testing, (4)
MLPA is not a suitable method to detect unknown point mutations, (5) MLPA probes are sensitive to small deletions, insertions, and mismatches, and (6) MLPA requires a CE analyzer,
which is a higher cost option compared with slab gel electrophoresis for RFLP.
Quantitative PCR
qPCR is a multiplex qPCR that coamplifies (in the same
tube) multiple genomic loci to determine gene copy number.18
The multiplex can consist of competitive coamplification of
SMN1, SMN2, the SMN internal standard, calibration factor,
and the calibration factor internal standard. In the competitive
PCR method, a known number of copies of a synthetic mutated
internal standard are introduced with the patient sample into the
PCR mixture. The internal standards are designed to be amplified with the same primer pairs for the SMN1 copy, with
efficiencies similar to those of the genomic DNA counterparts
and yield PCR products slightly smaller than the SMN PCR
product. The copy number of SMN1 is determined by coamplification of SMN1, SMN2, and the internal standards, and the
ratios are quantitated. End point detection of amplification of
fluorescently tagged PCR products is done by running the
samples through a CE analyzer. The major advantage of this
technique is that the internal standard is amplified with the same
primers that amplify the target sequence. Thus, the efficiency of
the amplification of the patient DNA and the internal standard
DNA should be very similar and allow one to accurately determine the gene copy number. Alternatively, real-time detection45
(as opposed to end point detection) of multiplex PCR reactions
using hydrolysis or hybridization fluorescent probes uses other
strategies for normalization including (1) standard curve
method or (2) comparative threshold method. Using the standard curve method, a standard curve is first constructed from a
DNA of known copy number. This curve is then used as a
reference standard for extrapolating quantitative information for
the SMN1 copy number. The cycle at which the curve crosses a
specified threshold is called the cycle threshold (Ct). Variation
introduced due to variable DNA inputs can be corrected by
normalizing to the calibration factor. Using the comparative
threshold method, the Ct values of the sample of interest are
compared with the Ct values of the calibration factor. Ct values
of both the calibrator and the sample of interest are normalized
to an appropriate endogenous internal control gene. qPCR is
able to detect copy number of specific genomic loci and, therefore, can be used to test for SMA diagnostic and carrier status.
Key advantages of qPCR include (1) extreme sensitivity, allowing the detection of less than five copies (perhaps only one copy
in some cases) of a target sequence, making it possible to
analyze small samples such as single cell analysis for the
purpose of preimplantation genetic diagnosis, (2) with appropriate internal standards and calculations, mean variation coefficients are 5–10%, allowing reproducible analysis of the gene
copy number, (3) all real-time platforms are relatively quick,
with some affording high-throughput automation, and (4) realtime platforms are performed in a closed reaction vessel that
requires no post-PCR manipulations, thereby minimizing the
chances for cross contamination in the laboratory. Important
limitations for qPCR techniques include (1) compounds present
in certain biological samples or sample collection compounds
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(i.e., heparin) may inhibit PCR, (2) DNA sequence variants
located under the primer binding sites of primers may reduce
proper annealing and might result in a false-positive carrier (one
copy) or diagnostic (zero copies) result, and (3) improper assay
development and incorrect data analysis. Unwarranted conclusions may present the largest limitation of qPCR, and therefore,
a robust and extensive validation is warranted to ensure specificity and accuracy of the results. Amplification and melting
curves must be visually inspected, whereas independent calculations based on these curves should be double checked for
accuracy. Finally, neither the MLPA nor qPCR can determine
whether two SMN1 genes are in cis on a single chromosome.
DNA sequencing
Although the absence of both copies of the SMN1 gene is a
very reliable and sensitive assay for the molecular diagnosis of
SMA, approximately 5% of affected patients have other types of
mutations in the SMN1 gene that will not be detected by
homozygous deletion testing. Because of the high deletion
frequency and according to the Hardy-Weinberg equilibrium,
most of these patients will be compound heterozygotes; with
one SMN1 allele being deleted and the other allele with a point
mutation or other types of small mutations. If a patient with a
SMA-like phenotype possesses only a single copy of SMN1, it
is likely that the remaining copy contains a more subtle mutation, including nonsense mutations, missense mutations, splice
site mutation insertions, and small deletions. The development
of high-throughput DNA sequencing techniques has made direct DNA sequencing of PCR-amplified genomic DNA a rapid
and economical approach to the identification of sequence mutations. As a consequence of the SMN1 gene being relatively
small and given the uniform spectrum of mutations, it is a
relatively straightforward procedure to sequence the gene and
identify mutations in patients who are negative for the diagnostic deletion test. However, it is necessary to verify that the
intragenic mutation has occurred in the SMN1 gene and not the
SMN2 gene. As an initial screen, primers that do not distinguish
between SMN1 and SMN2 may be used to amplify each exon for
direct DNA sequencing. If variants or mutations are identified,
SMN1-specific long-range PCR amplification is followed by
either direct DNA sequencing of that long-range product or
nested PCR sequencing.46 Important limitations for SMN direct
gene sequencing include (1) the requirement of allele-specific
sequencing of all variants identified, (2) DNA sequencing does
not detect large deletions or insertions, (3) mutations in patients
exhibiting mosaicism or chromosomal rearrangements may not
be detectable using sequencing technology, and (4) variants of
unknown significance.
INTERPRETATIONS
Elements considered essential to the reporting of clinical test
results are described in detail in the current ACMG Standards
and Guidelines for Clinical Genetics Laboratories. Examples of
model laboratory reports are included in the appendix. The
following elements must also be included in the reporting of
SMA results.
The methodology used to assign the SMA genotype should
be clearly stated. All positive results in clinically diagnosed/
suspected individuals should state that genetic counseling is
indicated, and carrier testing is available for other at-risk family
members.
Comments on phenotype, if included, should be abstract rather
than case specific. Although the inverse relationship between the
SMN2 copy number and disease severity has been well established,
© 2011 Lippincott Williams & Wilkins
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the report should clearly state that the relationship is not absolute if
reporting SMN2 levels. It is important that couples undergoing
carrier screening recognize that the carrier test does not provide
genotype/phenotype information. Type I SMA occurs in approximately 60% of the cases, whereas the milder types II and III
account for the remaining 40% of the cases.
Alternative diagnosis may be included when two normal
copies of the SMN1 gene have been detected. Other motor
neuron disorders should be considered such as SMA with respiratory distress, X-linked SMA, distal muscular atrophy, and
juvenile amyotrophic lateral sclerosis.
It is imperative that individuals understand the limitations of
the carrier test: two SMN1 genes in cis on the one chromosome
5, presence of rare de novo mutations, and the nondeletion
mutations. The issue of these false-negative results must be
included on all negative carrier reports. As is true for carrier
screening programs, the testing must be voluntary, and assurance of confidentiality is absolutely necessary.
Informed consent and the usual caveats should be addressed
including paternity issues, possible diagnostic errors due to
sample mix-ups, and genotype errors due the presence of rare
polymorphisms.
The following statement must be included on the report:
“This test was developed and its performance characteristics
determined by this laboratory. It has not been cleared or approved by the US Food and Drug Administration (FDA). The
FDA has determined that such clearance or approval is not
necessary. This analysis is used for clinical purposes. It should
not be regarded as investigational or for research.”
ACKNOWLEDGMENTS
The development of this laboratory practice guideline was
supported by an educational Grant from the Claire Altman
Heine Foundation, Inc., to the American College of Medical
Genetics Foundation.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Pearn J. Incidence, prevalence, and gene frequency studies of chronic
childhood spinal muscular atrophy. J Med Genet 1978;15:409 – 413.
Munstat TL, Davies KE. International SMA consortium meeting. Neuromuscular Disord 1992;2:423– 428.
Zerres K, Rudnik-Schoneborn S. Natural history in proximal spinal muscular
atrophy: clinical analysis of 445 patients and suggestions for a modification
of existing classifications. Arch Neurol 1995;52:518 –523.
Meldrum C, Scott C, Swoboda KJ. Spinal muscular atrophy genetic counseling access and genetic knowledge: parents perspective. J Child Neurol
2007;22:1019 –1026.
Lefebvre S, Burglen L, Reboullet S, et al. Identification and characterization
of a spinal muscular atrophy-determining gene. Cell 1995;80:155–165.
Burglen L, Lefebvre S, Clermont O, et al. Structure and organization of the
human survival motor neuron (SMN) gene. Genomics 1996;32:479 – 482.
Monani UR, McPherson JD, Burghes AH. Promoter analysis of the human
centromeric and telomeric survival motor neuron genes (SMNC and SMNT).
Biochim Biophys Acta 2007;1445:330 –336.
Echaniz-Laguna A, Miniou P, Bartholdi D, Melki J. The promoters of the
survival motor neuron gene (SMN) and its copy (SMNc) share common
regulatory elements. Am J Hum Genet 1999;64:1354 –1370.
Lefebvre S, Burlet P, Liu Q, et al. Correlation between severity and SMN
protein level in spinal muscular atrophy. Nat Genet 1997;16:265–269.
Coovert DD, Le TT, McAndrew PE, et al. The survival motor neuron protein
in spinal muscular atrophy. Hum Mol Genet 1997;6:1205–1214.
Paushkin S, Gubitz AK, Massenet S, Dreyfuss G. The SMN complex, an
assemblyosome of ribonucleoproteins. Curr Opin Cell Biol 2002;14:305–
312.
Yong J, Wan L, Dreyfuss G. Why do cells need an assembly machine for
RNA-protein complexes? Trends Cell Biol 2004;14:226 –232.
Monani UR. Spinal muscular atrophy: a deficiency in a ubiquitous protein;
a motor neuron-specific disease. Neuron 2005;48:885– 896.
Zhang HL, Pan F, Hong D, Shenoy SM, Singer RH, Bassell GJ. Active
transport of the survival motor neuron protein and the role of exon-7 in
cytoplasmic localization. J Neurosci 2003;23:6627– 6637.
Genetics
IN
Medicine • Volume 13, Number 7, July 2011
SMA standards and guidelines
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
Rossoll W, Jablonka S, Andreassi C, et al. SMN, the spinal muscular
atrophy-determining gene product, modulates axon growth and localization
of beta-actin mRNA in growth cones of motoneurons. J Cell Biol 2003;163:
801– 812.
Wirth B. An update of the mutation spectrum of the survival motor neuron
gene (SMN1) in autosomal recessive spinal muscular atrophy. Hum Mutat
2000;15:228 –237.
Campbell L, Potter A, Ignatius J, Dubowitz V, Davies K. Genomic variation
and gene conversion in spinal muscular atrophy: implications for disease
process and clinical phenotype. Am J Hum Genet 1997;61:40 –50.
McAndrew PE, Parsons DW, Simard LR, et al. Identification of proximal
spinal muscular atrophy carriers and patients by analysis of SMNT and
SMNC gene copy number. Am J Hum Genet 1997;60:1411–1422.
Wirth B, Herz M, Wetter A, et al. Quantitative analysis of survival motor
neuron copies: identification of subtle SMN1 mutations in patients with
spinal muscular atrophy, genotype-phenotype correlation, and implications
for genetic counseling. Am J Hum Genet 1999;64:1340 –1356.
Mailman MD, Heinz JW, Papp AC, et al. Molecular analysis of spinal
muscular atrophy and modification of the phenotype by SMN2. Genet Med
2002;4:20 –26.
Prior TW, Swoboda KJ, Scott HD, Hejmanowski AQ. Homozygous SMN1
deletions in unaffected family members and modification of the phenotype
by SMN2. Am J Med Genet A 2004;130:207–310.
Hsieh-Li HM, Chang JG, Jong YJ, et al. A mouse model for spinal muscular
atrophy. Nat Genet 2000;26:66 –70.
Monani UR, Coovert DD, Burghes AH. Animal models of spinal muscular
atrophy. Hum Mol Genet 2000;9:2451–2457.
Hoffman Y, Lorson CL, Stamm S, Androphy EJ, Wirth B. Htra2-beta 1
stimulates an exonic splicing enhancer and can restore full-length SMN
expression to survival motor neuron 2 (SMN2). Proc Natl Acad Sci USA
2000;97:9618 –9623.
Oprea GE, Krober S, McWhorter ML, et al. Plastin 3 is a protective
modifier of autosomal recessive spinal muscular atrophy. Science 2008;
320:524 –527.
Lorson CL, Androphy EJ. An exonic enhancer is required for inclusion of an
essential exon in the SMA-determining gene SMN. Hum Mol Genet 2000;
9:259 –265.
Lorson CL, Hahnen E, Androphy EJ, Wirth B. A single nucleotide in the
SMN gene regulates splicing and is responsible for spinal muscular atrophy.
Proc Natl Acad Sci USA 1999;96:6307– 6311.
Cartegni L, Kraniner AR. Disruption of an SF2/ASF-dependent exonic
splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of
SMN1. Nat Genet 2002;30:377–384.
Kashima T, Manley JL. A negative element in SMN2 exon 7 inhibits splicing
in spinal muscular atrophy. Nat Genet 2003;34:460 – 463.
Gavrilov DK, Shi X, Das K, Gilliam TC, Wang CH. Differential SMN2
expression associated with SMA severity. Nat Genet 1998;20:230 –231.
Prior TW, Krainer AR, Hua Y, et al. A positive modifier of spinal muscular
atrophy in the SMN2 gene. Am J Hum Genet 2009;85:408 – 413.
Alias L, Bernal S, Fuentes-Prior P, et al. Mutation update of spinal muscular
atrophy in Spain: molecular characterization of 745 unrelated patients and
identification of four novel mutations in the SMN1 gene. Hum Genet 2009;
125:29 –39.
Parsons DW, McAndrew PE, Iannaccone ST, et al. Intragenic telSMN
mutations: frequency, distribution, evidence of a founder effect and modification of spinal muscular atrophy phenotype by cenSMN copy number.
Am J Hum Genet 1998;63:1712–1723.
Hendrickson BC, Donohoe C, Akmaev VR, et al. Differences in SMN1 allele
frequencies among ethnic groups within North America. J Med Genet
2009;46:641– 644.
Ogino S, Wilson RB. Genetic testing and risk assessment for spinal muscular
atrophy (SMA). Hum Genet 2002;111:477–500.
Ogino S, Wilson RB, Gold B. New insights on the evolution of the SMN1
and SMN2 region: simulation and meta-analysis for allele and haplotype
frequency calculations. Eur J Hum Genet 2004;12:1015–1023.
Eggermann T. A new splice site mutation in the SMN1 gene causes discrepant results in SMN1 deletion screening approaches. Neuromusc dis 2008;18:
146 –149.
Wirth B, Schmidt T, Hahnen E, et al. De novo rearrangements found in 2%
of index patients with spinal muscular atrophy: mutational mechanisms,
parental origin, mutation rate, and implications for genetic counseling. Am J
Hum Genet 1997;61:1102–1111.
Prior TW. Carrier screening for spinal muscular atrophy. Genet Med 2008;
10:840 – 842.
ACOG Committee Opinion. Spinal muscular atrophy. Obstet Gynecol 2009;
113:1194 –1196.
Smith M, Calabro V, Chong B, et al. Population screening and cascade
testing for carriers of SMA. Eur J Hum Genet 2007;15:759 –766.
Prior TW. Perspectives and diagnostic considerations in spinal muscular
atrophy. Genet Med 2010;12:145–152.
van der Steege G, Grootscholten P, van der Vlies, et al. PCR-based DNA test
693
Genetics
Prior et al.
to confirm clinical diagnosis of autosomal recessive spinal muscular atrophy.
Lancet 1995;345:985–986.
44. Huang C-H, Chang YY, Chen CH. Copy number analysis of survival motor
neuron genes by multiplex ligation-dependent probe amplification. Genet
Med 2007;9:241–248.
45. Anhuf D, Eggermann T, Rudnik-Schoneborn S, Zerres K. Determination of
SMN1 and SMN2 copy number using Taqman technology. Hum Mutat
2003;22:74 –78.
46. Cheng S, Fockler C, Barnes WM, Higuchi R. Effective amplification of long
targets from cloned inserts and human genomic DNA. Proc Natl Acad Sci
USA 1994;91:5695–5699.
APPENDIX: MODEL LABORATORY REPORTS
Indication
Screening/carrier test/negative family history.
Reported ethnicity
White.
Comment
SMA is an autosomal recessive disease of variable age of
onset and severity caused by mutations (most often deletions
or gene conversions) in the SMN1 gene. Molecular testing
assesses the number of copies of the SMN1 gene. Affected
individuals have 0 copies of the SMN1 gene. Individuals with
one copy of the SMN1 gene are predicted to be carriers of
SMA. Individuals with two or more copies have a reduced
risk to be carriers. This copy number analysis cannot detect
individuals who are carriers of SMA as a result of either two
(or very rarely three) copies of the SMN1 gene on one
chromosome and the absence of the SMN1 gene on the other
chromosome or small intragenic mutations within the SMN1
gene. This analysis also will not detect germline mosaicism
or mutations in genes other than SMN1. Additionally, de
novo mutations have been reported in approximately 2% of
patients with SMA.
Methods
Specimen DNA is isolated and amplified by real-time PCR for
exon 7 of the SMN1 gene and two reference genes. A mathematical
algorithm is used to calculate the number of copies of SMN1.
Sequencing of the primer and probe binding sites for the SMN1
real-time PCR reaction is performed on all fetal samples and on
samples from individuals with one copy of SMN1 on carrier
testing, to rule out the presence of sequence variants, which could
interfere with analysis and interpretation. This test was developed
and its performance characteristics determined by this laboratory. It
has not been cleared or approved by the FDA. The FDA has
determined that such clearance or approval is not necessary. This
analysis is used for clinical purposes. It should not be regarded as
investigational or for research.
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Carrier frequency and risk reductions for individuals
with no family history of SMA
Ethnicity
Detection A priori
rate1 carrier risk34
Reduced carrier Reduced carrier
risk for 2
risk for 3
copy result
copy result
White
94.9%
1:35
1:632
1:3,500
Ashkenazi
Jewish
90.2%
1:41
1:350
1:4,000
Asian
92.6%
1:53
1:628
1:5,000
Hispanic
90.6%
1:117
1:1,061
1:11,000
African
American
71.1%
1:66
1:121
1:3,000
Mixed
ethnicities
For counseling purposes, consider using the ethnic
background with the most conservative risk
estimates.
General disclaimer
False-positive or -negative results may occur for reasons that
include genetic variants, blood transfusions, bone marrow transplantation, erroneous representation of family relationships, or
contamination of a fetal sample with maternal cells.
Example of a negative SMA carrier report
Result
SMN1 copy number: 2 (reduced carrier risk).
Interpretation
This individual has an SMN1 copy number of 2. This result
reduces but does not eliminate the risk to be a carrier of SMA.
Ethnic-specific risk reductions based on negative family history
and an SMN1 copy number of 2 are provided in the comment
section. Genetic counseling is recommended.
Example of a positive SMA carrier report
Result
SMN copy number: 1 (carrier).
Interpretation
This individual has one copy of SMN1 and is, therefore,
predicted to be a carrier of SMA, a disease of variable age of
onset and severity. Genetic counseling is recommended.
© 2011 Lippincott Williams & Wilkins